Whole-genome Identification of the Perilla frutescens ARF Transcription Factor Family and Functional Analysis of Its Role in Abiotic Stress Responses

doi:10.13560/j.cnki.biotech.bull.1985.2025-0877

Abstract

Abstract:

Objective Auxin response factor (ARF) plays a crucial regulatory role in plant hormone signal transduction and abiotic stress responses. The genome-wide identification of the Perilla frutescens PfARF transcription factor (TF) family and the analysis of the expression characteristics of related genes under abiotic stress provide a theoretical basis for elucidating the function of PfARF in the stress response of perilla. Method The PfARF members were identified from the genome of perilla, and their chromosomal location, phylogenetic evolution and conserved structures were analyzed using omic tools, such as TBtools, MEGA11, NCBI-CD Search, et al. The expressions of PfARF genes in the developing seeds of perilla and in the seedlings under abiotic stress was analyzed through RNA-seq and RT-qPCR. The PfARF22 gene was obtained using molecular cloning technology, and the subcellular localization of its encoded protein was detected by transient transformation in Nicotiana benthamiana leaves. A protein interaction network diagram of the PfARF22 TF was constructed, and its binding sites, binding energies, and other parameters were analyzed using molecular docking technology. Result A total of 29 PfARF family members were identified in perilla, which were classified into four subgroups. PfARF members in the same subgroup exhibited similarities in their conserved motifs and gene structures, and segment duplication was the main driving force for their evolution and expansion. PfARF genes showed differential expression in the developing seeds of perilla and in the abiotic stress conditions of the seedlings. The expression of PfARF22 gene significantly increased under low temperature and drought stress conditions. Subcellular localization analysis revealed that PfARF22 was located in nucleus. The molecular docking results indicated that PfARF22 and PfMADS1 cooperatively regulated biological processes such as the growth and development of perilla and responses to abiotic stress. Conclusion Perilla PfARF TF family consists of 29 members. PfARF22 has potential regulatory effects on the growth and development of perilla as well as responses to stress conditions such as low temperature and drought.

Key words: Perilla frutescens, auxin response factor, bioinformatics analysis, expression characteristic analysis, abiotic stress, groth and development

HUANG Xu-sheng, ZHOU Ya-li, JIA Zheng-rong, SHEN Shan, CHEN Jin-chai, ZHOU Guang-li, LI Xin-xin, WANG Ji-ping, LI Run-zhi, JIA Xiao-yun. Whole-genome Identification of the Perilla frutescens ARF Transcription Factor Family and Functional Analysis of Its Role in Abiotic Stress Responses[J]. Biotechnology Bulletin, 2026, 42(7): 148-162.

Figures/Tables 11

Fig. 1 Chromosome distribution of PfARF genes in P. frutescens

Fig. 2 Phylogenetic tree of ARF proteins in P. frutescens

Fig. 3 Conserved motif pattern and gene structure analysis of PfARF members in P. frutescensA: Evolutionary relationship of PfARF members. B: Protein functional domain. C: Conserved motif pattern. D: Gene structure

Fig. 4 Synteny analysis of inter-chromosomal relationships of PfARF genes in P. frutescens

Fig. 5 Collinearity relationship of ARF genes among P. frutescens, A. thaliana and S. indicumThe blue lines indicate the collinearity relationship of ARF genes between P. frutescens and A. thaliana; the pink lines indicate the collinearity relationship of ARF genes between P. frutescens and S. indicum

Fig. 6 Cis-acting regulatory elements predicted in the promoters of PfARF genes

Fig. 7 Expression analysis of PfARF genes at different stages of seed development in P. frutescens10, 20, and 30 DAF indicate the seeds of perilla at 10, 20, and 30 d after flowering, respectively

Fig. 8 Analysis of phenotype (A) and PfARF genes expression (B) of P. frutescens seedlings under low temperature stressThe data are the mean values ± standard errors (mean ± SE) of three biological replicates. Different lowercase letters indicate significant differences between samples (P<0.05, Tukey’s multiple comparison method). The same below

Fig. 9 Analysis of phenotype (A) and PfARF genes expression (B) of P. frutescens seedlings under drought stress

Fig. 10 Cloning of PfARF22 gene (A) and subcellular localization of PfARF22 protein (B) in P. frutescens

Fig. 11 Analysis of PfARF22 protein interaction network (A) and molecular docking (B‒F) of P. frutescens

References 39

[1]	安艳, 欧巧明, 王丽, 等. 紫苏叶活性化学成分研究进展及其抗氧化与抑菌活性的物质基础 [J]. 中国农学通报, 2025, 41(20): 128-142.
	An Y, Ou QM, Wang L, et al. Research progress on active chemical components of perilla leaves and their antioxidant and antibacterial activity [J]. Chin Agric Sci Bull, 2025, 41(20): 128-142.
[2]	Park YJ, Dixit A, Ma KH, et al. Evaluation of genetic diversity and relationships within an on-farm collection of Perilla frutescens (L.) Britt. using microsatellite markers [J]. Genet Resour Crop Evol, 2008, 55(4): 523-535.
[3]	Guan LJ, Zhu L, Zhang XD, et al. Perilla seed oil and protein: composition, health benefits, and potential applications in functional foods [J]. Molecules, 2024, 29(22): 5258.
[4]	Kaur S, Godara S, Singh N, et al. Multivariate data analysis assisted mining of nutri-rich genotypes from north eastern Himalayan germplasm collection of perilla (Perilla frutescens L.) [J]. Plant Foods Hum Nutr, 2024, 79(4): 843-850.
[5]	Li BJ, Bao RX, Shi YN, et al. Auxin response factors: important keys for understanding regulatory mechanisms of fleshy fruit development and ripening [J]. Hortic Res, 2024, 11(10): uhae209.
[6]	Li YH, Han S, Qi YH. Advances in structure and function of auxin response factor in plants [J]. J Integr Plant Biol, 2023, 65(3): 617-632.
[7]	Guilfoyle TJ, Hagen G. Auxin response factors [J]. Curr Opin Plant Biol, 2007, 10(5): 453-460.
[8]	Shen CJ, Yue RQ, Sun T, et al. Genome-wide identification and expression analysis of auxin response factor gene family in Medicago truncatula [J]. Front Plant Sci, 2015, 6: 73.
[9]	江海洋, 魏巍, 刘艳, 等. 高粱生长素反应因子(ARF)基因的全基因组分析与进化研究 [J]. 安徽农业大学学报, 2010, 37(3): 395-400.
	Jiang HY, Wei W, Liu Y, et al. Genome-wide analysis and evolution of the auxin response factor (ARF) gene family in Sorghum bicolor [J]. J Anhui Agric Univ, 2010, 37(3): 395-400.
[10]	Ha CV, Le DT, Nishiyama R, et al. The auxin response factor transcription factor family in soybean: genome-wide identification and expression analyses during development and water stress [J]. DNA Res, 2013, 20(5): 511-524.
[11]	Zouine M, Fu YY, Chateigner-Boutin AL, et al. Characterization of the tomato ARF gene family uncovers a multi-levels post-transcriptional regulation including alternative splicing [J]. PLoS One, 2014, 9(1): e84203.
[12]	Wang YJ, Deng DX, Shi YT, et al. Diversification, phylogeny and evolution of auxin response factor (ARF) family: insights gained from analyzing maize ARF genes [J]. Mol Biol Rep, 2012, 39(3): 2401-2415.
[13]	Pernisová M, Vernoux T. Auxin does the SAMba: auxin signaling in the shoot apical meristem [J]. Cold Spring Harb Perspect Biol, 2021, 13(12): a039925.
[14]	Okushima Y, Fukaki H, Onoda M, et al. ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis [J]. Plant Cell, 2007, 19(1): 118-130.
[15]	Zheng Y, Zhang K, Guo L, et al. AUXIN RESPONSE FACTOR3 plays distinct role during early flower development [J]. Plant Signal Behav, 2018, 13(5): e1467690.
[16]	Wang CF, Li XM, Zhuang YB, et al. A novel miR160a-GmARF16-GmMYC2 module determines soybean salt tolerance and adaptation [J]. New Phytol, 2024, 241(5): 2176-2192.
[17]	Hu W, Zuo J, Hou XW, et al. The auxin response factor gene family in banana: genome-wide identification and expression analyses during development, ripening, and abiotic stress [J]. Front Plant Sci, 2015, 6: 742.
[18]	黄旭升, 周雅莉, 史先飞, 等. 紫苏bZIP转录因子全基因组鉴定及对非生物胁迫的响应分析 [J]. 植物生理学报, 2023, 59(7): 1383-1397.
	Huang XS, Zhou YL, Shi XF, et al. Genome-wide characterization and abiotic stress response analysis of bZIP transcription factors in Perilla frutescens [J]. Plant Physiol J, 2023, 59(7): 1383-1397.
[19]	Zhang YJ, Shen Q, Leng L, et al. Incipient diploidization of the medicinal plant perilla within 10,000 years [J]. Nat Commun, 2021, 12(1): 5508.
[20]	Wang DK, Pei KM, Fu YP, et al. Genome-wide analysis of the auxin response factors (ARF) gene family in rice (Oryza sativa) [J]. Gene, 2007, 394(1/2): 13-24.
[21]	刘萌萌. 转录因子GhAHL4负调控GhSDP1参与棉籽萌发过程油脂降解 [D]. 太谷: 山西农业大学, 2022.
	Liu MM. Characterization of GhAHL4, a transcription factor that negatively regulates the triacylglycerol lipase of GhSDP1 in Gossypium hirsutum [D]. Taigu: Shanxi Agricultural University, 2022.
[22]	Zhang HH, Li LL, He YQ, et al. Distinct modes of manipulation of rice auxin response factor OsARF17 by different plant RNA viruses for infection [J]. Proc Natl Acad Sci USA, 2020, 117(16): 9112-9121.
[23]	Guo F, Huang YZ, Qi PP, et al. Functional analysis of auxin receptor OsTIR1/OsAFB family members in rice grain yield, tillering, plant height, root system, germination, and auxinic herbicide resistance [J]. New Phytol, 2021, 229(5): 2676-2692.
[24]	李慧峰, 冉昆, 何平, 等. 苹果生长素响应因子(ARF)基因家族全基因组鉴定及表达分析 [J]. 植物生理学报, 2015, 51(7): 1045-1054.
	Li HF, Ran K, He P, et al. Genome-wide identification and expression analysis of auxin response factor (ARF) gene family in apple [J]. Plant Physiol J, 2015, 51(7): 1045-1054.
[25]	Yoko Okushima PJO. Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19 [J]. Plant Cell, 2005, 17(2): 444-463.
[26]	Wang HM, Huang HT, Shang YK, et al. Identification and characterization of auxin response factor (ARF) family members involved in fig (Ficus carica L.) fruit development [J]. PeerJ, 2022, 10: e13798.
[27]	田时雨, 张蓓林, 雷阳, 等. 茶树全基因组生长素反应因子(ARF)基因家族鉴定及表达分析 [J]. 中国茶叶, 2022, 44(6): 37-46.
	Tian SY, Zhang BL, Lei Y, et al. Identification and expression analysis of whole genome auxin response factor (ARF) gene family in tea plant [J]. China Tea, 2022, 44(6): 37-46.
[28]	Tang YH, Bao XX, Liu K, et al. Genome-wide identification and expression profiling of the auxin response factor (ARF) gene family in physic nut [J]. PLoS One, 2018, 13(8): e0201024.
[29]	马杰茹, 武小娟, 王沛捷, 等. 彩色马铃薯ARF基因家族的鉴定与表达分析 [J]. 中国农业大学学报, 2025, 30(7): 105-118.
	Ma JR, Wu XJ, Wang PJ, et al. Screening and expression analysis of ARF gene related to anthocyanin synthesis in colored potatos [J]. J China Agric Univ, 2025, 30(7): 105-118.
[30]	秦文俊, 熊言杰, 赵冉, 等. 甘蓝ARF基因家族的鉴定及其在非生物胁迫下的表达分析 [J]. 生物技术通报, 2025, 41(10): 253-263.
	Qin WJ, Xiong YJ, Zhao R, et al. Identification and expression analysis of the ARF gene family in cabbage under abiotic stress [J]. Biotechnol Bull, 2025, 41(10): 253-263.
[31]	王占军, 李静静, 周子寒, 等. 油桐ARF基因家族的鉴定及表达分析 [J]. 江苏农业学报, 2024, 40(6): 984-992.
	Wang ZJ, Li JJ, Zhou ZH, et al. Identification and expression analysis of ARF gene family in Vernicia fordii [J]. Jiangsu J Agric Sci, 2024, 40(6): 984-992.
[32]	黄贤斌, 张太奎, 刘翠玉, 等. 石榴ARF基因家族鉴定及表达分析 [J]. 果树学报, 2019, 36(1): 43-55.
	Huang XB, Zhang TK, Liu CY, et al. Identification and expression analysis of ARF gene family in pomegranate [J]. J Fruit Sci, 2019, 36(1): 43-55.
[33]	孟思达, 聂晓雨, 叶云珠, 等. 植物ARF家族成员研究进展 [J]. 沈阳农业大学学报, 2025, 56(2): 160-168.
	Meng SD, Nie XY, Ye YZ, et al. Research progress on members of the plant ARF family [J]. J Shenyang Agric Univ, 2025, 56(2): 160-168.
[34]	胡晓, 侯旭, 袁雪, 等. ARF和Aux/IAA调控果实发育成熟机制研究进展 [J]. 生物技术通报, 2017, 33(12): 37-44.
	Hu X, Hou X, Yuan X, et al. Research progress on mechanism of ARF and Aux/IAA regulating fruit development and ripening [J]. Biotechnol Bull, 2017, 33(12): 37-44.
[35]	曹丽茹, 叶飞宇, 李伟亚, 等. 玉米生长素响应因子基因ZmARF10在干旱胁迫中的功能分析 [J]. 华北农学报, 2024, 39(2): 1-10.
	Cao LR, Ye FY, Li WY, et al. Functional analysis of maize auxin response factor gene ZmARF10 under drought stress [J]. Acta Agric Boreali Sin, 2024, 39(2): 1-10.
[36]	刘江, 黄盖群, 代洁, 等. 桑树ARF基因家族的鉴定与表达分析 [J]. 蚕业科学, 2023, 49(3): 193-200.
	Liu J, Huang GQ, Dai J, et al. Identification and expression analysis of ARF gene family in mulberry [J]. Acta Sericologica Sin, 2023, 49(3): 193-200.
[37]	李秉贞. 生长素响应因子ARF4通过与IAA12互作调控拟南芥芽再生 [D]. 泰安: 山东农业大学, 2020.
	Li BZ. ARF4 regulates shoot regeneration through interaction with IAA12 in Arabidopsis [D]. Tai’an: Shandong Agricultural University, 2020.
[38]	Wang Y, Li Y, He SP, et al. The transcription factor ERF108 interacts with AUXIN RESPONSE FACTORs to mediate cotton fiber secondary cell wall biosynthesis [J]. Plant Cell, 2023, 35(11): 4133-4154.
[39]	Hu JH, Li X, Sun TP. Four class A AUXIN RESPONSE FACTORs promote tomato fruit growth despite suppressing fruit set [J]. Nat Plants, 2023, 9(5): 706-719.